The first complete mitogenome of the endangered spotted eagle ray, Aetobatus ocellatus, reinforces its classification within the family Aetobatidae

43 The complete mitochondrial genome of the endangered spotted eagle ray Aetobatus ocellatus 44 was determined for the first time using Next Generation Sequencing (NGS) reads mined from 45 the Sequence Read Archive (SRA) of the GenBank database (BioSample SAMN31811701, 46 collection site: Bohol Island, Philippines ). The spotted eagle ray mitogenome (GenBa nk 47 accession pending ) has 20,217 bp in length displaying a typical vertebrate mitogenome 48 organization (13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region). 49 A phylogenetic analysis based on the nucleotide sequence information of 13 protein-coding 50 genes from different related species confirmed the species status of A. ocellatus as an Indo -51 West Pacific species , which was formerly described as A. narinari whose current accepted 52 distribution is restricted to the Atlantic Ocean. 53 54 55 56

Aetobatidae, mitogenome, phylogenetics, Next Generation Sequencing 57 58 59 60 61 62 63 64 65 66 67 68 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 15, 2025. ; https://doi.org/10.1101/2025.01.13.632509doi: bioRxiv preprint Mitogenome Announcement 3 Eagle r ays of the genus Aetobatus (Myliobatiformes: Aetobatidae) suffer from strong 69 overfishing and slow recovery from overexploitation due to their low regeneration rate, slow 70 growth, and late maturity ( Yamaguchi et al. 2021), which has led to a continued population 71 decline throughout their distribution ranges (Dulvy et al. 2021; Finucci et al. 2024) and 72 subsequent categorization of all of its members as Endangered or Vulnerable within the IUCN 73 red list (IUCN 2024). The classification of Aetobatidae species is particularly susceptible to 74 taxonomic confusion due to the interspecific overlap of their morphological features (White et 75 al. 2013) and during the last decade a number of taxonomic changes were introduced with the 76 aid of genetic markers, including the resurrection of old synonyms and description of new 77 species (White 2010; White and Moore 2013; White et al. 2013). 78 Herein, I report the first complete mitochondrial genome of the endangered spotted eagle ray 79 Aetobatus ocellatus, which has only recently been resurrected as the valid name for the Indo -80 West Pacific member of the narinari complex (White et al. 2010). The A. ocellatus sample was 81 collected in Bohol Island, Philippines (BioSample: SAMN31811701) and sequenced by the 82 Smithsonian National Museum of Natural History in their “NOAA Genome Skimming of 83 Marine animals” project (BioProject accession: PRJNA720393). Paired-end FASTQ genomic 84 reads (2.2 Gbp within 14.6 million reads, Platform: Illumina NovaSeq 6000) were downloaded 85 from the public GenBank SRA repository (SRA accession: SRS15852991), trimmed with 86 BBDuk (14.5 million reads remained), and merged with BBMerge (4.1 million reads remained) 87 implemented in Geneious Prime v. 2021.1.1 (Biomatters Ltd., Auckland, New Zealand) . 88 Unmerged (22,239) and merged (16,553) reads were independently assembled against the only 89 complete available mitogenome reference sequence of Aetobatus prior to this study (A. 90 flagellum, 20,201 bp, GenBank accession NC_02283 7) using the “Map to Reference” tool in 91 Geneious Prime. Protein-coding (PC), transport RNA (tRNA), and ribosomal RNA (rRNA) 92 genes were annotated using the “Annotate and Predict” feature of Geneious Prime by 93 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 15, 2025. ; https://doi.org/10.1101/2025.01.13.632509doi: bioRxiv preprint Mitogenome Announcement 4 comparing the novel A. ocellatus mitogenome to other complete mitogenomes of closely 94 related species. The position of tRNAs was verified using tRNAscan -SE (Chan and Lowe 95 2019). 96 The 20,217 bp consensus mitogenome of A. ocellatus (GenBank accession pending) has a 97 typical fish mitogenome organization (Winn et al. 2024) with 13 PC, two rRNA, 22 tRNA 98 genes, and one putative control region, showing an overall nucleotide composition of A: 31.4%, 99 C: 27.1%, G: 15.5%, and T: 26%. All PCGs use the typical ATG start codon except for the 100 COI which utilizes the alternative GTG start codon. 101 The Bayesian phylogenetic analysis was performed using MrBayes 3.2 (Ronquist & 102 Huelsenbeck 2003) and consisted of a concatenated matrix of 13 PCGs (11,460 bp) from 15 103 mitogenomes of closely related species retrieved from the Gen Bank database. Substitution 104 saturation in single codon positions from each PCG was analyzed using DAMBE 5 (Xia 2013). 105 The software jModelTest 2 (Darriba et al. 2012) was used to find the best-fit model of evolution 106 for each PCG. Figur e 1 shows the Bayesian phylogenetic tree in which all samples were 107 grouped into 4 distinct family clades according to their taxonomy. Aetobatidae species formed 108 a basal monophyletic clade with robust statistical support. A. ocellatus was clustered together 109 with A. narinari into a discrete subclade (338 variable sites, genetic divergence 3.03%) , 110 reflecting their low estimated divergence age (from 2 to 5 mya, Sales et al. 2019). The genetic 111

analyzed herein (BioSample SAMN31811701) came from an organism collected in 112 the Indo-West Pacific region ( Bohol Island, Philippines) originally identified as A. narinari. 113 However, the phylogenetic results obtained herein indicate that BioSample SAMN31811701 114 belongs to A. ocellatus, supporting its species status as an Indo-West Pacific species (White et 115 al. 2010), while A. narinari is restricted to the Atlantic region (Sales et al. 2019). 116 117 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 15, 2025. ; https://doi.org/10.1101/2025.01.13.632509doi: bioRxiv preprint Mitogenome Announcement 5 Figure 1. Bayesian ph ylogenetic tree inferred from 13 concatenated mitochondrial protein -118 coding genes of Aetobatidae, Dasyatidae, Mobulidae, and Rhinopteridae species. The sequence 119 matrix (11,460 nt) used in the phylogenetic analysi s consisted of unambiguously aligned 120 regions of the first, second, and third codon positions. The position of Aetobatus ocellatus is 121 highlighted in bold. Pteroplatytrygon violacea and Dasyatis pastinaca (Dasyatidae) were used 122 as outgroups. Posterior probabilities are shown at each node. GenBank accession numbers for 123 each species are shown in parentheses. 124 125 126 127 128 129 DASYATIDAE (OUTGROUP) 0.05 Mobula alfredi (NC_068734.1) Mobula birostris (KX151648) Mobula mobular (KT203434.1) Mobula tarapacana (KX151647) Mobula eregoodootenke (NC_025954.1) Mobula kuhlii (KX151651.1) Mobula thurstoni (NC_037219.1) Mobula hypostoma (PQ760241.1) Mobula munkiana (KX151645.1) Rhinoptera brasiliensis (NC_068668.1) Rhinoptera bonasus (NC_082999.1) Aetobatus narinari (KX151649.1) Aetobatus ocellatus (PENDING) Aetobatus flagellum (NC_022837.1) Pteroplatytrygon violacea (KJ641617) Dasyatis pastinaca (NC_057976.1) MOBULIDAE RHINOPTERIDAE AETOBATIDAE 0.05 1 0.98 0.99 1 1 1 11 1 1 1 1 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 15, 2025. ; https://doi.org/10.1101/2025.01.13.632509doi: bioRxiv preprint Mitogenome Announcement 6

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